Reappraisal of Frequency of Common Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations in Iranian Cystic Fibrosis Patients.

Background
Cystic Fibrosis (CF) is a life-threatening recessive genetic disorder resulting from mutations in the gene encoding the fibrosis transmembrane conductance regulator protein (CFTR). The CF clinical phenotype shows wide variation ranging from severe disease in early childhood in those homozygous for the p.Phe508del mutation to absence of the vas deferens in otherwise healthy men homozygous for the p.Arg117His mutation.


Materials and Methods
DNA was extracted from whole blood from 62 patients with CF. The CFTR mutation was determined by Allele-Specific PCR assay. The spearman and linear regression analysis were used to obtain the correlation between phenotype and genotype relationship.


Results
Out of total 62 patients, 35 (56.4%) were male. The mean age of the patients was 15.56 ± 6.65 years. Mutations in CFTR were detected in 64.5% of the patients. The commonest mutations were p.Phe508del (33.9%), p.Arg117His; [5T] (5.64%), p.Arg117His; [7T] (4.03%) and p.Trp1282X (5.64%). Mutations p.Ile507del (4%), p.Gly542X (4%), p.Asn1303Lys (2.42%), c.489+1G>T (1.6%), p.Gly551Asp (1.6%) and c.1585-1G>A (1.6%) were also detected. Most mutations were detected in west and south of Iran, while p.Phe508del mutation was dominant mutation (75%) in east and southeast of Iran. The study showed either an association between this mutation with severity of disease and sex or an association between p.Arg117His mutations and age at diagnosis.


Conclusion
The geographic distribution of gene mutation in Iranian cystic fibrosis patients was very heterogenic. In spite of the study that showed a correlation between p.Phe508del and severity of disease, to find any correlation between genotype and phenotype a broad and multi-centered study is recommended.


INTRODUCTION
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes the CFTR protein (1). More than 2000 CFTR mutations are associated with abnormal function of the chloride channel (2). Mutation frequencies vary between different ethnic populations based on their geographic distribution (3). Published data suggest that the frequency and distribution of CFTR mutations vary in Iranian populations depending on their geographical region and ethnicity (2,(4)(5)(6)(7).
Generally, the loss of function or expression of CFTR causes severe symptoms of Cystic Fibrosis (CF), but mutations that reduce function or expression of CFTR cause atypical and mild form of disease (8). However, different studies do not always agree on the relationship between genotype and phenotype in CF (8,9).
A comprehensive study to identify CFTR mutations across several ethnic populations is very important and should be considered in health planning. For this reason, this study was conducted to recognize the common 11 CFTR gene mutations as well as poly T polymorphisms in 62 CF patients who were registered from the different geographical regions of Iran at the Pediatric Respiratory Disease Research Center which serves as a national referral CF center. Many studies on imaging, clinical, and paraclinical aspects as well as mortality of disease have been done in this center (10)(11)(12)(13). Also the correlation of genotype and phenotype of the disease was assessed in these patients.

DNA extraction
Genomic DNA was extracted from two ml of whole blood stored in Na-EDTA using a silicon-based extraction kit according to manufacturer instruction (YTZ, IRAN).
The concentration and quality of DNA was evaluated by spectrophotometer at 260 and 280 nm, and aliquots stored both at 4°C for immediate use or -20°C for long term storage.

Demographic analysis
In this study 62 CF patients registered at the Pediatric

Genotype analysis
In total, we found a mutation in at least one allele of  ) and Enterococcus was also more common in them. Our results showed that the most frequent mutation in Iran was p.Phe508del. A (33.9%). The frequency of the p.Phe508del mutation in this cohort was higher than previous reports in Iran (4,5,7,17), but lower than those reported in European countries and higher than eastern neighboring countries such as Pakistan and India (18).
It seems that Iran acts as a bridge between these regions. We have also shown that the frequency of Since our center is a national referral center for pediatric respiratory disease, we serve a wide population from all regions of Iran, and most of our patients are referred with severe respiratory signs and symptoms of the CF, such as bronchiectasis (12). We think this is the most likely reason for the higher rate of p.Phe508del mutation in this group of patients. The second probable reason is the higher consanguinity rate in this study in comparison with similar studies in Iran (74.2 vs 60%) (4).
Our study showed that there was a significant difference between p.Phe508del mutation and gender of the patients (p<0.032; CI: 95%) which was more common in females. There was a positive correlation between these variables. Given that CFTR is located on an autosomal chromosome there is no clear explanation for this finding. in Iran neither did consider these mutations nor found any mutation in these codons (2,(4)(5)(6)(7)31).
None of the studied cases showed any mutation in p.Arg553X and p.Ar560Thr. The result was similar to previous studies conducted in Iran (2,(4)(5)(6)(7)31). According to the opinion of the authors, there is possible to find the specific mutations in these codons of CFTR gene in Iranian patients. So it shall not be omitted from screening program of CF.
Since about 35% of the CF patients did not carry the CFTR mutations we tested, it is possible to find rare and specific mutations in the Iranian population based on complete sequences of the gene. It seems that the whole genome sequencing can reveal some other and new specific mutation in Iranian patients. Unfortunately, we couldn't do that because of limitation of budget. But the whole genome sequencing of CFTR gene is recommended to find new mutations in this group of patients.

CONCLUSION
The study showed a wide heterogeneity of mutation in the CFTR gene within the CF patients registered at the Pediatric Respiratory Disease Research Center of Iran.
Based on p.Phe508del and p.Trp1282X mutations, it seems that Iranian CF patients are more similar to Arab and Sephardic Jews race than Ashkenazi Jews. Consequently, it is essential to design a national screening program. On the other hand, in order to find the relationship between genotype and phenotype in some codons and any logical correlation between the genotype and phenotype of disease in CF patients, an extensive and inclusive study has to be designed.